Effects of plant growth regulators on transient expression of foreign gene in Nicotiana benthamiana L. leaves.

BACKGROUND: In the last decades, replicating expression vectors based on plant geminivirus have been widely used for enhancing the efficiency of plant transient expression. By using the replicating expression vector derived from bean yellow dwarf virus and green fluorescent protein as a reporter, we investigated the effects of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine, as three common plant growth regulators, on the plant biomass and efficiency of transient expression during the process of transient expression in Nicotiana benthamiana L. leaves. RESULTS: With the increase of the concentration of α-naphthalene acetic acid, gibberellins3, and 6-benzyladenine (from 0.1 to 1.6 mg/L), the fresh weight, dry weight, and leaf area of the seedlings increased first and then returned to the levels similar to the controls (without chemical treatment). The treatment with α-naphthalene acetic acid at 0.2 and 0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.4 mg/L α-naphthalene acetic acid and was increased about by 19%, compared to the controls. Gibberellins3 at 0.1-0.4 mg/L can enhance the level of transient expression of green fluorescent protein, which peaked at 0.2 mg/L gibberellins3 and was increased by 25%. However, the application of 6-benzyladenine led to decrease in the level of transient expression of green fluorescent protein. CONCLUSIONS: The appropriate plant growth regulators at moderate concentration could be beneficial to the expression of foreign genes from the Agrobacterium-mediated transient expression system in plants. Thus, appropriate plant growth regulators could be considered as exogenous components that are applied for the production of recombinant protein by plant-based transient expression systems.

[Calibration Transfer of Near Infrared Spectrometric Models for Crude Protein of Protein Feed Materials].

The near infrared spectrometric quantitative model of protein feed and its sharing in different instruments can greatly improve the utilization efficiency of the model and meet the needs of rapid development of feed industry. Considering the issue of applicability of near infrared spectrometric models for crude protein of protein feed materials, calibration transfer was explored among three types of instruments using spectral subtraction correction, direct standardization and piecewise directs standardization methods for the first time. Four kinds of protein feed raw materials were involved in the present study, corn protein powder, rapeseed meal, fish meal and distillers dried grains with soluble. The experimental instruments included MATRIX-I Fourier transform near infrared instrument (master instrument), Spectrum 400 Fourier transform near infrared instrument (slave 1 instrument), and SupNIR-2750 grating near infrared instrument (slave 2 instrument). Results showed that the spectral data difference for all the samples between the master and slave 2 instrument was relatively small, and the difference between the master and slave 1 instrument, and slave 1 and slave 2 instrument were relatively large. All the root mean square error of prediction and bias values after calibration transfer were lower than the values before calibration transfer, except that no improvement was found for the prediction of corn protein powder of slave 2 instrument corrected by piecewise direct standardization method. The relative prediction deviation (RPD) of corn protein powder, rapeseed meal and distillers dried grains with soluble transferred by all three methods were higher than 3, which indicated good predictions, while the RPD of fish meal were all higher than 2.5, which indicated relative good predictions. All three techniques used in the study were effective in the correction of the difference between different instruments for protein feed materials. This study is of important practical significance for the application of near infrared spectrometric models for crude protein of protein feed materials.

Combined application of plasma mutagenesis and gene engineering leads to 5-oxomilbemycins A3/A4 as main components from Streptomyces bingchenggensis.

Milbemycin oxime has been commercialized as effective anthelmintics in the fields of animal health, agriculture, and human infections. Currently, milbemycin oxime is synthesized by a two-step chemical reaction, which involves the ketonization of milbemycins A3/A4 to yield the intermediates 5-oxomilbemycins A3/A4 using CrO3 as catalyst. Due to the low efficiency and environmental unfriendliness of the ketonization of milbemycins A3/A4, it is imperative to develop alternative strategies to produce 5-oxomilbemycins A3/A4. In this study, the atmospheric and room temperature plasma (ARTP) mutation system was first employed to treat milbemycin-producing strain Streptomyces bingchenggensis, and a mutant strain BC-120-4 producing milbemycins A3, A4, B2, and B3 as main components was obtained, which favors the construction of genetically engineered strains producing 5-oxomilbemycins. Importantly, the milbemycins A3/A4 yield of BC-120-4 reached 3,890 ± 52 g/l, which was approximately two times higher than that of the initial strain BC-109-6 (1,326 ± 37 g/l). The subsequent interruption of the gene milF encoding a C5-ketoreductase responsible for the ketonization of milbemycins led to strain BCJ60 (∆milF) with the production of 5-oxomilbemycins A3/A4 and the elimination of milbemycins A3, A4, B2, and B3. The high 5-oxomilbemycins A3/A4 yield (3,470 ± 147 g/l) and genetic stability of BCJ60 implied the potential use in industry to prepare 5-oxomilbemycins A3/A4 for the semisynthesis of milbemycins oxime.